Polymer-semaxanib moiety conjugates

ABSTRACT

The invention relates to (among other things) polymer-semaxanib moiety conjugates and related compounds. A compound of the invention, when administered by any of a number of administration routes, exhibits advantages over the semaxanib moiety in unconjugated form.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/910,830, filed Mar. 2, 2018, now allowed, which is adivisional application of U.S. patent application Ser. No. 13/995,301,filed Aug. 27, 2013, now U.S. Pat. No. 9,943,605, which is a 35 U.S.C. §371 application of International Application No. PCT/US2011/067175,filed Dec. 23, 2011, designating the United States, which claims thebenefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 61/426,880, filed Dec. 23, 2010, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD

This invention comprises (among other things) chemically modified formsof the receptor tyrosine kinase (RTK) inhibitor, semaxanib, which formspossess certain advantages over semaxanib lacking the chemicalmodification. The chemically modified forms of semaxanib and relatedforms of semaxanib described herein relate to and/or have application(s)in (among others) the fields of drug discovery, pharmacotherapy,physiology, organic chemistry and polymer chemistry.

BACKGROUND

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues withinproteins. Phosphorylation of these hydroxy groups is required for thegrowth, differentiation and proliferation of cells. Thus, virtually allaspects of the cell life cycle depend on normal PK activity. In view ofthe criticality normal PK activity has on healthy cell functioning, itis perhaps not surprising that abnormal PK activity has been related toa host of disorders, ranging from relatively non-life threateningdiseases such as psoriasis to extremely virulent diseases such asglioblastoma (brain cancer).

Among other categorizations, PKs can be divided into two classes, thecytoplasmic protein tyrosine kinases (PTKs) and the transmembranereceptor tyrosine kinases (RTKs). Briefly, the RTKs comprise a family oftransmembrane receptors with diverse biological activity. The HERsubfamily of RTKs includes EGFR (epithelial growth factor receptor),HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylatedligand binding domain, a transmembrane domain and an intracellularcytoplasmic catalytic domain that can phosphorylate tyrosine residues onproteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated alpha subunitsand two beta subunits which cross the cell membrane and which containthe tyrosine kinase domain.

A third RTK subfamily is referred to as the “platelet derived growthfactor receptor” (“PDGF-R”) group, which includes PDGF-R-α, PDGF-R-β,CSFI-R, c-kit and c-fms. These receptors consist of glycosylatedextracellular domains composed of variable numbers of immunoglobin-likeloops and an intracellular domain wherein the tyrosine kinase domain isinterrupted by unrelated amino acid sequences.

Another group, which, because of its similarity to the PDGF-R subfamily(and is sometimes subsumed into the PDGF-R subfamily) is the fetus liverkinase (“flk”) receptor subfamily. This group is believed to be made upof kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1,VEGF-R2), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

Semaxanib is a potent and selective inhibitor of the Flk-1/KDR vascularendothelial growth factor (VEGF) receptor tyrosine kinase. It targetsthe VEGF pathway, and both in vivo and in vitro studies havedemonstrated its antiangiogenic potential. Chemically, sunitinib'ssystematic name is“(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1,3-dihydro-2H-indol-2-one”and formula is provided below.

Although initial testing with semaxanib was encouraging, a clinicaltrial associated with semaxanib showed that the drug did not achievecertain defined endpoints.

Therefore, a need exists to provide compounds that can exert the samepharmacology semaxanib in vivo, yet have properties different fromsemaxanib. The present invention seeks to address this and/or otherneeds.

SUMMARY

In one or more embodiments of the invention, a compound is provided, thecompound comprising a semaxanib moiety residue covalently attached via aspacer moiety (e.g., a releasable linkage-containing spacer moiety) to awater-soluble, non-peptidic oligomer.

In one or more embodiments of the invention a compound is provided, thecompound having the following

wherein:

-   -   R¹ is selected from the group consisting of H and halo (e.g.,        F);    -   R² is selected from the group consisting of H, halo (e.g., F or        Cl), NO₂ and lower alkyl (e.g., CH₃);    -   R³ is selected from the group consisting of H and lower alkyl        (e.g., CH₃);    -   R⁴ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (e.g., CH₃ and CH₂CH₃) and CH₂CH₂COOH;    -   R⁵ is selected from the group consisting of H, lower alkyl        (e.g., CH₃ and CH₂CH₃), COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃;    -   R⁶ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (CH₃, SCH₃);    -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety); and    -   POLY¹ is a first water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention a compound is provided, thecompound having the following

wherein:

-   -   R¹ is selected from the group consisting of H and halo (e.g.,        F);    -   R² is selected from the group consisting of H, halo (e.g., F or        Cl), NO₂ and lower alkyl (e.g., CH₃);    -   R³ is selected from the group consisting of H and lower alkyl        (e.g., CH₃);    -   R⁴ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (e.g., CH₃ and CH₂CH₃) and CH₂CH₂COOH;    -   R⁵ is selected from the group consisting of H, lower alkyl        (e.g., CH₃ and CH₂CH₃), COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃;    -   R⁶ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (CH₃, SCH₃),    -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety); and    -   POLY¹ is a first water-soluble, non-peptidic polymer, and        and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a compound is provided, thecompound having the following structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety); and    -   POLY¹ is a first water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention (containing two differentattachment points for a water-soluble, non-peptidic polymer), a compoundis provided, the compound having the following structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety);    -   POLY¹ is a first water-soluble, non-peptidic polymer;    -   Xr is a releasable linkage-containing spacer moiety; and    -   POLY² is a second water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention (containing two differentattachment points for a water-soluble, non-peptidic polymer), a compoundis provided, the compound having the following structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety);    -   POLY¹ is a first water-soluble, non-peptidic polymer;    -   Xr is a releasable linkage-containing spacer moiety; and    -   POLY is a water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof.

In one or more embodiments of the invention, a composition is provided,the composition comprising (i) a compound comprising a semaxanib moietyresidue covalently attached via a releasable linkage-containing spacermoiety to a water-soluble, non-peptidic polymer, and, optionally, (ii) apharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound as described herein, wherein thecompound is present in a dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicpolymer to a semaxanib moiety.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound as described herein to amammal in need thereof.

Additional embodiments of the present conjugates, compositions, methods,and the like will be apparent from the following description, examples,and claims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the mean plasma concentration values of sunitiniband mPEG₃₋₉-semaxanib compounds following 2 mg/kg intravenous dosing inrats, as further discussed in Example 4.

FIG. 2 is a plot of the mean plasma concentration values of sunitiniband mPEG₃₋₉-semaxanib compounds following 2 mg/kg oral dosing in rats,as further discussed in Example 4.

FIG. 3 is a plot of the mean plasma concentration values of sunitiniband mPEG₃₋₉-semaxanib compounds following 20 mg/kg oral dosing in rats,as further discussed in Example 4.

DETAILED DESCRIPTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic polymer” indicates an polymer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” polymer transmits at least 75%, more preferably at least95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-soluble polymeris at least 95% (by weight) soluble in water or completely soluble inwater. With respect to being “non-peptidic,” a polymer is non-peptidicwhen it has less than 35% (by weight) of amino acid residues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer. In the case of a homo-polymer, a single repeating structuralunit forms the polymer. In the case of a co-polymer, two or morestructural units are repeated—either in a pattern or randomly—to formthe polymer. Preferred polymers used in connection with present theinvention are homo-polymers. The water-soluble, non-peptidic polymercomprises one or more monomers serially attached to form a chain ofmonomers. The polymer can be formed from a single monomer type (i.e., ishomo-polymeric) or two or three monomer types (i.e., is co-polymeric).

A “polymer” is a molecule possessing from about 2 to about 2000 or more(e.g., 2000 to 20000) monomers. Specific oligomers for use in theinvention include those having a variety of geometries such as linear,branched, or forked, to be described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG polymer” or any polyethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though, the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG polymers for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG polymers, thevariable (n) ranges from about 2 to 2000 or more (e.g., 2000 to 20000),and the terminal groups and architecture of the overall PEG can vary.When PEG further comprises a functional group, A, for linking to, e.g.,a small molecule drug, the functional group when covalently attached toa PEG oligomer does not result in formation of an oxygen-oxygen bond(—O—O—, a peroxide linkage).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. In addition, the end-capping group may contain a targeting moiety.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises a vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell-specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, quantum dots, and the like.

“Branched,” in reference to the geometry or overall structure of apolymer, refers to a polymer having two or more polymers “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of apolymer, refers to a polymer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer branches or forks from a linear structure intoone or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “releaseable linkage” is a relatively labile bond that cleaves underphysiological conditions. An exemplary releasable linkage is ahydrolyzable bond that cleaves upon reaction with water (i.e., ishydrolyzed). The tendency of a bond to hydrolyze in water may depend notonly on the general type of linkage connecting two atoms but also on thesubstituents attached to these atoms. Appropriate hydrolyticallyunstable or weak linkages include but are not limited to carboxylateester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides, oligonucleotides, thioesters, andcarbonates. Another exemplary releasable linkage is an enzymaticallyreleasable linkage. An “enzymatically releasable linkage” means alinkage that is subject to cleavage by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced. An“alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, acyl, cyano. The substituted alkyl groups may besubstituted once, twice or three times with the same or with differentsubstituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 7 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention causes no significant adverse toxicological effects to apatient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthalenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g., 1-2, 1-3 or 1-4 substituents)chosen from halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a compound described herein that is neededto provide a desired level of active agent and/or conjugate in thebloodstream or in the target tissue. The precise amount may depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of the composition, intended patientpopulation, patient considerations, and may readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a semaxanib moiety residue covalentlyattached (either directly or through one or more atoms) to awater-soluble, non-peptidic polymer.

The semaxanib moiety residue is a residue of a semaxanib moiety. In oneor more embodiments of the invention, the semaxanib moiety isencompassed by the following structure:

wherein:

-   -   R¹ is selected from the group consisting of H and halo (e.g.,        F);    -   R² is selected from the group consisting of H, halo (e.g., F or        Cl), NO₂ and lower alkyl (e.g., CH₃)    -   R³ is selected from the group consisting of H and lower alkyl        (e.g., CH₃);    -   R⁴ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (e.g., CH₃ and CH₂CH₃) and CH₂CH₂COOH.    -   R⁵ is selected from the group consisting of H, lower alkyl        (e.g., CH₃ and CH₂CH₃), COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; and    -   R⁶ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (CH₃, SCH₃).

An exemplary semaxanib moiety is semaxanib (also referred to as“SU5416”), the chemical structure of which is provided above.

Semaxanib and other semaxanib moieties can be prepared in accordancewith the procedures set forth in this disclosure as well as in Sun etal. (1998) J. Med. Chem. 41:2588-2603 and in U.S. Pat. Nos. 6,147,106,6,610,688, 6,906,093, 6,908,930 and 7,202,265.

Exemplary compounds of the invention are encompassed by the followingstructure:

wherein:

-   -   R¹ is selected from the group consisting of H and halo (e.g.,        F);    -   R² is selected from the group consisting of H, halo (e.g., F or        Cl), NO₂ and lower alkyl (e.g., CH₃);    -   R³ is selected from the group consisting of H and lower alkyl        (e.g., CH₃);    -   R⁴ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (e.g., CH₃ and CH₂CH₃) and CH₂CH₂COOH;

R⁵ is selected from the group consisting of H, lower alkyl (e.g., CH₃and CH₂CH₃), COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; and

-   -   R⁶ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (CH₃, SCH₃),        and pharmaceutically acceptable salts thereof. It is understood        that, with respect to the generic structure provided in Formula        I-C, attachment of “˜X-POLY¹” will take place at one of the R⁴,        R⁵ and R⁶ positions. Thus, for example, Formula I-Ca provides a        generic structure in which attachment of “˜X-POLY¹” occurs at        the R⁵ location.

Further exemplary compounds of the invention are encompassed by thefollowing structure:

wherein:

-   -   R¹ is selected from the group consisting of H and halo (e.g.,        F);    -   R² is selected from the group consisting of H, halo (e.g., F or        Cl), NO₂ and lower alkyl (e.g., CH₃);    -   R³ is selected from the group consisting of H and lower alkyl        (e.g., CH₃);    -   R⁴ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (e.g., CH₃ and CH₂CH₃) and CH₂CH₂COOH;    -   R⁵ is selected from the group consisting of H, lower alkyl        (e.g., CH₃ and CH₂CH₃), COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; and    -   R⁶ is selected from the group consisting of H, halo (e.g., Br),        lower alkyl (CH₃, SCH₃),        and pharmaceutically acceptable salts thereof. It is understood        that, with respect to the generic structure provided in Formula        II-C, attachment of “˜X-POLY¹” will take place at one of the        available ring positions (e.g., R¹, R² and R³ positions).

Still further exemplary compounds of the invention are encompassed bythe following structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety); and    -   POLY¹ is a first water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof.

Additional exemplary compounds of the invention are encompassed by thefollowing structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety);    -   POLY¹ is a first water-soluble, non-peptidic polymer;    -   Xr is a releasable linkage-containing spacer moiety; and    -   POLY² is a second water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof. Approaches for        providing attachment of ˜Xr-POLY² can be found in U.S.        Provisional Patent Application No. 61/426,919, filed on Dec. 23,        2010, and entitled “Polymer-Sunitinib Conjugates” (and the        international patent application claiming priority thereto        having the same title and filed on Dec. 23, 2011) and in U.S.        Provisional Patent Application No. 61/426,893, filed on Dec. 23,        2010, and entitled “Polymer-Des-ethyl Sunitinib Conjugates” (and        the international patent application claiming priority thereto        having the same title and filed on Dec. 23, 2011).

Still more additional exemplary compounds of the invention areencompassed by the following structure:

wherein:

-   -   X is spacer moiety (which can be stable or be a releasable        linkage-containing spacer moiety);    -   POLY¹ is a first water-soluble, non-peptidic polymer;    -   Xr is a releasable linkage-containing spacer moiety; and    -   POLY is a water-soluble, non-peptidic polymer,        and pharmaceutically acceptable salts thereof. Approaches for        providing attachment of ˜Xr-POLY² can be found in U.S.        Provisional Patent Application No. 61/426,919, filed on Dec. 23,        2010, and entitled “Polymer-Sunitinib Conjugates” (and the        international patent application claiming priority thereto        having the same title and filed on Dec. 23, 2011) and in U.S.        Provisional Patent Application No. 61/426,893, filed on Dec. 23,        2010, and entitled “Polymer-Des-ethyl Sunitinib Conjugates” (and        the international patent application claiming priority thereto        having the same title and filed on Dec. 23, 2011).

The Spacer Moiety

The spacer moiety (the linker through which the water-soluble,non-peptidic polymer is attached to the semaxanib moiety) may be asingle bond, a single atom, such as an oxygen atom or a sulfur atom, twoatoms, or a number of atoms. A spacer moiety is typically but is notnecessarily linear in nature. In one or more embodiments, the spacermoiety, “X,” is stable (and comprises no releasable linkages). In one ormore embodiments, the spacer moiety, “X,” comprises a releasablelinkage. Preferably, the spacer moiety “X” is one having a chain lengthof less than about 12 atoms, and preferably less than about 10 atoms,and even more preferably less than about 8 atoms and even morepreferably less than about 5 atoms, whereby length is meant the numberof atoms in a single chain, not counting substituents. For instance, aurea linkage such as this, R_(oligomer)—NH—(C═O)—NH—R′_(drug,) isconsidered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selectedembodiments, the linkage does not comprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) other atoms,as described further below.

More specifically, in selected embodiments, a spacer moiety, “X,” may beany of the following: “—” (i.e., a covalent bond, that may be stable ordegradable, between the substituted aromatic triazine residue and thewater-soluble, non-peptidic oligomer), —O—, —NH—, —S—, —C(O)—, —C(O)O—,—OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, C(O)—NH,NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂,—CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—,—CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group, —N(R⁶)—, R⁶ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl. Additional spacer moieties include, acylamino, acyl,aryloxy, alkylene bridge containing between 1 and 5 inclusive carbonatoms, alkylamino, dialkylamino having about 2 to 4 inclusive carbonatoms, piperidino, pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino,1-piperizinyl, 4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-loweralkyl)-1-piperizinyl, 4-(methoxy-lower alkyl)-1-piperizinyl, andguanidine. In some instances, a portion or a functional group of thedrug compound may be modified or removed altogether to facilitateattachment of the oligomer. In some instances, it is preferred that X isnot an amide, i.e., —CONR— and —RNCO—).

For purposes of the present invention, however, a group of atoms is notconsidered a linkage when it is immediately adjacent to an oligomersegment, and the group of atoms is the same as a monomer of the oligomersuch that the group would represent a mere extension of the oligomerchain.

The spacer moiety “X” between the water-soluble, non-peptidic polymerand the semaxanib moiety is formed by reaction of a functional group ona terminus of the polymer (or nascent polymer when it is desired to“grow” the polymer onto the semaxanib moiety) with a correspondingfunctional group within the semaxanib moiety. Illustrative reactions aredescribed briefly below. For example, an amino group on a polymer may bereacted with a carboxylic acid or an activated carboxylic acidderivative on the semaxanib moiety, or vice versa, to produce an amidelinkage. Alternatively, reaction of an amine on a polymer with anactivated carbonate (e.g., succinimidyl or benzotriazolyl carbonate) onthe semaxanib moiety, or vice versa, forms a carbamate linkage. Reactionof an amine on a polymer with an isocyanate (R—N═C═O) on a semaxanib, orvice versa, forms a urea linkage (R—NH—(C═O)—NH—R′). Further, reactionof an alcohol (alkoxide) group on a polymer with an alkyl halide, orhalide group within a semaxanib moiety, or vice versa, forms an etherlinkage. In yet another coupling approach, a small molecule having analdehyde function is coupled to a polymer amino group by reductiveamination, resulting in formation of a secondary amine linkage betweenthe oligomer and the semaxanib.

A particularly preferred water-soluble, non-peptidic polymer is apolymer bearing an amine functional group. In this regard, the polymerwill have the following structure: CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—NH₂,wherein (n) is one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is one of1, 2, 3, 4, 5, 6 and 7. Preferred (n) values include 2, 3, 4 and 5 andpreferred (p) values include 2, 3 and 4.

In one or more embodiments of the invention, a spacer moiety containedin the molecule is a releasable linkage-containing spacer moiety (e.g.,“Xr” in each of Formulae II-C and III-C). A releasablelinkage-containing spacer moiety must be one that will cleave in vivofollowing administration to a patient. In this regard, releasablelinkages are known to those of ordinary skill in the art. In addition,whether a given linkage can serve as a releasable linkage in connectionwith the compounds provided herein can be tested through experimentation(e.g., by administering a compound having the proposed releasablelinkage to a patient and testing, e.g., via chromatographic techniques,periodically obtained blood samples for indications of cleavage).

Exemplary releasable linkages for use in connection with the compoundsprovided herein include, without limitation, thioether, carbamate,ester, carbonate, urea and enzyme-cleavable peptidic linkages.Thioether, carbamate, ester, carbonate, urea can cleave via a(β-elimination reaction (with or without the enzymatic coordination,e.g., an ester can serve as a releaseable linkage herein regardless ofwhether the ester will be cleaved via an esterase). With respect toenzyme-cleavable peptidic linkages, the spacer moiety can include aseries of amino acids known to be a substrate for an enzyme present inthe intended patient population. In this way, upon administration to thepatient, the enzyme-cleavable peptidic linkage-containing compound ofthe invention, will cleave the enzyme-cleavable peptidic linkage viaenzymatic cleavage, thereby releasing semaxanib (or semaxanib with arelatively small molecular fragment). Examples of peptidic linkagessubject to enzymatic cleavage in a given patient population have beendescribed (see, for example, U.S. Patent Application Publication No.2005/0079155) and can be determined experimentally.

In one or more embodiments of the invention, the releasablelinkage-containing spacer moiety, “Xr,” can take the followingstructure:

˜[X¹]_(a)-Lr-[X²]_(b)˜  (Formula III)

wherein:

-   -   (a) is either zero or one;    -   (b) is either zero or one;    -   X¹, when present, is a first spacer;    -   Lr is the releasable linkage; and    -   X², when present, is a second spacer.

In those instances of Formula III wherein both (a) and (b) are zero, itwill be understood that the releasable linkage-containing spacer moietyis made up of only the releasable linkage. That is, the releasablelinkage-containing spacer moiety only contains the releasable linkageand no other atoms are present between the semaxanib residue and thewater-soluble, non-peptidic polymer.

In those instances of Formula III wherein either or both of (a) and (b)are one, it will be understood that the releasable linkage-containingspacer moiety contains one or more additional atoms other than thosethat make up the releasable linkage. Nonlimiting exemplary spacers(e.g., X¹ and X²) that may flank the releasable linkage include —O—,—NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—,—C(O)O—CH₂—, —OC(O)—CH₂—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl. Additionalspacers include, acylamino, acyl, aryloxy, alkylene bridge containingbetween 1 and 5 inclusive carbon atoms, alkylamino, dialkylamino havingabout 2 to 4 inclusive carbon atoms, piperidino, pyrrolidino, N-(loweralkyl)-2-piperidyl, morpholino, 1-piperizinyl, 4-(loweralkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl,4-(methoxy-lower alkyl)-1-piperizinyl, fluorenyl, and guanidine. Forpurposes of the present invention, however, a group of atoms is notconsidered a spacer when it is immediately adjacent to an polymericsegment, and the group of atoms is the same as a monomer of the polymersuch that the group would represent a mere extension of the polymerchain.

When present, a spacer is typically but is not necessarily linear innature. In addition, a spacer is typically but is not necessarilyhydrolytically stable and/or is enzymatically stable. In one or moreembodiments of the invention, the spacer, when present, has a chainlength of less than about 12 atoms (e.g., less than about 10 atoms, lessthan about 8 atoms, and less than about 5 atoms). With respect todetermining length of a particular spacer, length herein is defined asthe number of atoms in a single chain, not counting substituents. Forinstance, a urea linkage such as this,R_(polymer)—NH—(C═O)—NH—R′_(drug,) is considered to have a chain lengthof three atoms (—NH—C(O)—NH—).

The Water-Soluble, Non-Peptidic Polymer, “POLY¹, “POLY²,” and so Forth

The compounds of the invention include a water-soluble, non-peptidicpolymer. A wade array of polymers can be used and the invention is notlimited with respect to the type (e.g., polyethylene oxide,polyoxazoline, and so forth), size (e.g., from 2 to 4000 monomers insize) and geometry (e.g., linear, branched, multi-armed, and so forth)used.

With respect to type, the water-soluble, non-peptidic polymer can beunderstood as a series of repeating monomers, wherein the type ofmonomer(s) dictates the type of water-soluble, non-peptidic polymer.Exemplary monomers include, but are not limited to the group consistingof: alkylene oxides, such as ethylene oxide or propylene oxide; olefinicalcohols, such as vinyl alcohol, 1-propenol or 2-propenol; vinylpyrrolidone; hydroxyalkyl methacrylamide and hydroxyalkyl methacrylate,where, in each case, alkyl is preferably methyl; α-hydroxy acids, suchas lactic acid or glycolic acid; phosphazene, oxazoline, carbohydratessuch as monosaccharides, alditol such as mannitol; andN-acryloylmorpholine. In one or more embodiments, the water-soluble,non-peptidic polymer is a co-polymer of two monomer types selected fromthis group, or, more preferably, is a homo-polymer of one monomer typeselected from this group. With respect to co-polymers, the two monomertypes in a co-oligomer may be of the same monomer type, for example, twoalkylene oxides, such as ethylene oxide and propylene oxide.

With respect to size, the water-soluble, non-peptidic polymer can be arelatively small or the water-soluble, non-peptidic polymer can berelatively large.

In those embodiments in which a relatively small water-soluble,non-peptidic polymer is present, exemplary values of molecular weightsinclude: below about 2000; below about 1500; below about 1450; belowabout 1400; below about 1350; below about 1300; below about 1250; belowabout 1200; below about 1150; below about 1100; below about 1050; belowabout 1000; below about 950; below about 900; below about 850; belowabout 800; below about 750; below about 700; below about 650; belowabout 600; below about 550; below about 500; below about 450; belowabout 400; below about 350; below about 300; below about 250; belowabout 200; and below about 100 Daltons. Exemplary ranges for arelatively small water-soluble, non-peptidic polymer include from about100 to about 1400 Daltons; from about 100 to about 1200 Daltons; fromabout 100 to about 800 Daltons; from about 100 to about 500 Daltons;from about 100 to about 400 Daltons; from about 200 to about 500Daltons; from about 200 to about 400 Daltons; from about 75 to 1000Daltons; and from about 75 to about 750 Daltons.

For relatively small water-soluble, non-peptidic polymers, the number ofmonomers in will typically fall within one or more of the followingranges: between 1 and about 30 (inclusive); between about 2 and about25; between about 2 and about 20; between about 2 and about 15; betweenabout 2 and about 12; between about 2 and about 10. In certaininstances, the number of monomers in series in the polymer (and thecorresponding conjugate) is one of 1, 2, 3, 4, 5, 6, 7, or 8. Inadditional embodiments, the polymer (and the corresponding conjugate)contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers. Inyet further embodiments, the polymer (and the corresponding conjugate)possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series.Thus, for example, when the water-soluble, non-peptidic polymer includesCH₃—(OCH₂CH₂)_(n)—, “n” is an integer that can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30, and can fall within one or more of the followingranges: between about 1 and about 25; between about 1 and about 20;between about 1 and about 15; between about 1 and about 12; betweenabout 1 and about 10.

When the water-soluble, non-peptidic polymer has 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 monomers, these values correspond to a methoxy end-cappedpoly(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When thepolymer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped poly(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the molecular weight of the water-soluble, non-peptidic polymer inthe compound is relatively large (e.g., greater than 2,000 Daltons), theweight can fall within the range of 2,000 Daltons to about 150,000Daltons. Exemplary ranges, however, include molecular weights in therange of from about 3,000 Daltons to about 120,000 Daltons; in the rangeof from about 5,000 Daltons to about 110,000 Daltons; in the range offrom greater than 5,000 Daltons to about 100,000 Daltons, in the rangeof from about 6,000 Daltons to about 90,000 Daltons, in the range offrom about 10,000 Daltons to about 85,000 Daltons, in the range ofgreater than 10,000 Daltons to about 85,000 Daltons, in the range offrom about 20,000 Daltons to about 85,000 Daltons, in the range of fromabout 53,000 Daltons to about 85,000 Daltons, in the range of from about25,000 Daltons to about 120,000 Daltons, in the range of from about29,000 Daltons to about 120,000 Daltons, in the range of from about35,000 Daltons to about 120,000 Daltons, and in the range of from about40,000 Daltons to about 120,000 Daltons.

Exemplary molecular weights for relatively large water-soluble,non-peptidic polymers include about 2,200 Daltons, about 2,500 Daltons,about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons,about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000Daltons, and about 75,000 Daltons. Branched versions of thewater-soluble, non-peptidic polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) andmulti-arm versions of the water-soluble, non-peptidic polymer (e.g., afour-armed 40,000 Dalton water-soluble polymer comprised of four 10,000Dalton polymers) having a total molecular weight of any of the foregoingcan also be used.

Thus, regardless of whether a relatively small or large water-soluble,non-peptidic polymer is used, when the water-soluble, non-peptidicpolymer is a poly(ethylene oxide), the polymer will comprise a number of(OCH₂CH₂) monomers [or (CH₂CH₂O) monomers, depending on how the PEG isdefined]. As used throughout the description, the number of repeatingunits is identified by the subscript “n” in “(OCH₂CH₂)_(n).” Thus, thevalue of (n) typically falls within one or more of the following ranges:from 2 to about 3400, from about 100 to about 2300, from about 100 toabout 2270, from about 136 to about 2050, from about 225 to about 1930,from about 450 to about 1930, from about 1200 to about 1930, from about568 to about 2727, from about 660 to about 2730, from about 795 to about2730, from about 795 to about 2730, from about 909 to about 2730, andfrom about 1,200 to about 1,900. For any given polymer in which themolecular weight is known, it is possible to determine the number ofrepeating units (i.e., “n”) by dividing the total weight-averagemolecular weight of the polymer by the molecular weight of the repeatingmonomer.

With respect to geometry, any geometry (e.g., linear, branched,multi-armed) can be used in connection with the conjugates of theinvention and the invention is not limited in this regard.

With respect to linear water-soluble, non-peptidic polymers, typically,although not necessarily, a linear water-soluble, non-peptidic polymerwill be terminally end capped with a substantially inert group (e.g.,with a methyl or methoxy group) on the terminus not attached toreleasable linkage-containing spacer moiety. In one or more embodiments,however, compounds of invention having a linear, water-soluble,non-peptidic polymer will not be terminally end capped with asubstantially inert group and will instead have a functional group. Insuch embodiments, the linear, water-soluble, non-peptidic polymer canafford compounds of the invention having two semaxanib moiety residuesattached to it. In another form of such embodiments, the linear,water-soluble, non-peptidic polymer can afford compounds of theinvention having a single semaxanib moiety residue and a residue of adifferent moiety (e.g., a targeting moiety).

With respect to branched water-soluble, non-peptidic polymers, thesepolymers typically contain a two discernable end capped water-soluble,non-peptidic polymers connected via a branch point, which is connectedthrough a spacer to either a functional group (prior to conjugation) orsemaxanib moiety residue. Exemplary branched forms of water-soluble,non-peptidic polymers are described herein and in WO 2005/107815, WO2005/108463, U.S. Pat. Nos. 5,932,462 and 7,026,440, and U.S. PatentApplication Publication No. 2005/0009988. Among other benefits, branchedwater-soluble, non-peptidic polymers—given the presence of twodiscernable water-soluble, non-peptidic polymers—have the potential toprovide greater polymer character compared to, for example, a linearpolymer having a single water-soluble, non-peptidic polymer.

As used herein, reference to a “water-soluble, non-peptidic polymer”(e.g., “POLY”) is considered to include branched and multi-arm formseven though two or more discernable water-soluble, non-peptidic polymerscan be identified.

With respect to multi-arm water-soluble, non-peptidic polymers, thesepolymers typically contain three or more discernable water-soluble,non-peptidic polymers, each having the ability to covalently attach to amoiety of interest, and each typically connected to a central coremoiety (e.g., a residue of a polyol). Among other benefits, multi-armwater-soluble, non-peptidic polymers—given the ability of each arm tocovalently attach to a drug—have the potential to provide greater drugcharacter compared to, for example, a linear polymer having a singledrug attached thereto.

Methods for Synthesizing Compounds of the Invention

The compounds discussed herein can be prepared in a variety of methodsand the invention is not limited in this regard.

In one or more embodiments, the compounds of the prepared by a methodcomprising covalently attaching a water-soluble, non-peptidic polymer toa semaxanib moiety.

With respect to the water-soluble, non-peptidic polymer, such polymerscan be obtained commercially in a form bearing one or more reactivegroups, thereby providing a reagent suited for facile covalentattachment to the semaxanib moiety. In this form, the water-soluble,non-peptidic polymer is sometimes conventionally referred to as apolymeric reagent. Commercial suppliers for polymeric reagents includeSigma-Aldrich (St. Louis, Mo.), Creative PEGWorks (Winston Salem, N.C.USA), SunBio PEG-Shop (SunBio USA, Orinda, Calif.), JenKem TechnologyUSA (Allen, Tex.), and NOF America Corporation (White Plains, N.Y.).Using routine experimentation, one of ordinary skill in the art canidentify polymeric reagents having suitable sizes, geometries, andreactive groups and so forth for preparing the compounds of theinvention. For example, it is possible to prepare a series of compoundswherein each member in the series differs in a feature (e.g., the sizeof the water-soluble, non-peptidic polymer, the type of reactive group,the ability of a linkage to release, and so forth) and then administerone member in the series to a patient followed by periodic detection andquantification (e.g., using chromatographic techniques) of the compound(and/or metabolite thereof) in blood and/or urine samples along with theassessment of a clinical endpoint. Each member of the series isadministered, quantified and assessed in a similar way to a naïvepatient. Once each members of the series is tested, the results can bereviewed to determine which feature(s) provided compounds having thedesired effect(s).

Covalently attaching the polymeric reagent to the semaxanib moiety istypically conducted under conjugation conditions, which conditionsinclude combining the semaxanib moiety with a polymeric reagent (often amolar excess of polymeric reagent relative to the semaxanib moiety)under conditions of temperature, pH, time and solvent that allow forcovalent attachment between a reactive group of the polymeric reagent tothe semaxanib moiety.

Exemplary conjugation conditions between a given polymeric reagentbearing a reactive group and the semaxanib moiety will be known to oneof ordinary skill in the art based upon the disclosure provided hereinand in the context of the relevant literature. See, for example,Poly(ethylene glycol) Chemistry and Biological Applications, AmericanChemical Society, Washington, D.C. (1997).

Exemplary linear, branched and multi-armed polymeric reagents (alongwith exemplary conjugation conditions for those polymeric reagents)along with the releasable linkage-containing compounds formed therefromare provided in U.S. Provisional Patent Application No. 61/426,919,filed on Dec. 23, 2011, and entitled “Polymer-Sunitinib Conjugates” andin U.S. Provisional Patent Application No. 61/426,893, filed on Dec. 23,2011, and entitled “Polymer-Des-ethyl Sunitinib Conjugates.” Utility andTesting of Compounds.”

Animal models (rodents and dogs) can used to study oral drug transport.In addition, non-in vivo methods include rodent everted gut excisedtissue and Caco-2 cell monolayer tissue-culture models. These models areuseful in predicting oral drug bioavailability (thereby providing anindication of whether a given compound of the invention can beadministered orally).

To test for binding activity, a compound can be tested using in vitrobinding studies to receptors using various cell lines expressing thesereceptors. In vitro binding studies known to those of ordinary skill inthe art can be used to test the binding for a receptor of interest.

The following assay may be used to determine the level of activity andeffect of a compound on protein kinases. The assay is performed in anELISA (Enzyme-Linked Immunosorbent Sandwich Assay) format [Voller et al.(1980), “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371]. The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound. Similar assays can bedesigned along the same lines for any protein kinase using techniqueswell known in the art.

Using this basic approach, one can test over 80 protein kinases,including GST-Flk1, pyk2, PYK2, FGFR-1R, EGFR, PDGFR, HER-2, CDK2, andIGF-1.

The compounds of the invention may be tested in animal models of cancersto determine their cancer-inhibition potential. An exemplary model isthe xenograft-based assay. In this assay, the ability of human tumors togrow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides auseful in vivo model for studying the biological response to therapiesfor human tumors. Since the first successful xenotransplantation ofhuman tumors into athymic mice [Rygaard et al. (1969) Acta Pathol.Microbial. Scand. 77:758-760), many different human tumor cell lines(e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck,glioblastoma, bone, and malignant melanomas) have been transplanted andsuccessfully grown in nude mice.

In addition to an approach as provided in the Experimental, thefollowing assays may be used to determine the level of activity,specificity and effect of the different compounds of the presentinvention. Three general types of assays are useful for evaluatingcompounds: cellular/catalytic, cellular/biological and in vivo. Theobject of the cellular/catalytic assays is to determine the effect of acompound on the ability of a tyrosine kinase to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a tyrosine kinase in a cell. The object of the invivo assays is to determine the effect of a compound in an animal modelof a particular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC #CCL 107), A375 cells (melanoma, ATCC #CRL 1619),A431 cells (epidermoid carcinoma, ATCC #CRL 1555), Calu 6 cells (lung,ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL 1435), SKOV3TP5 cells andNIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR,IGF-1R or any other test kinase. The following protocol can be used toperform xenograft experiments.

Female athymic mice (BALB/c, nu/nu) are maintained under clean-roomconditions in micro-isolator cages with Alpha-dri bedding. They receivesterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium [for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)]. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for ten minutes. Pellets are resuspended in sterilePBS or media (without FBS) to a particular concentration and the cellsare implanted into the hindflank of the mice (8-10 mice per group,2-10×10⁶ cells/animal). Tumor growth is measured over 3 to 6 weeks usingvenier calipers. Tumor volumes are calculated as a product oflength×width×height. P values are calculated using the Students t-test.Test compounds in 50-100 μL excipient (DMSO, or VPD:DSW) can bedelivered by IP injection at different concentrations generally startingat day one after implantation.

The compounds of the invention may be administered per se or in the formof a pharmaceutically acceptable salt, and any reference to thecompounds of the invention herein is intended to includepharmaceutically acceptable salts. If used, a salt of a compound asdescribed herein should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Such pharmacologically and pharmaceutically acceptable saltscan be prepared by reaction of the compound with an organic or inorganicacid, using standard methods detailed in the literature. Examples ofuseful salts include, but are not limited to, those prepared from thefollowing acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulfonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

The compounds of the invention may contain one or more chiral centersand for each chiral center, the invention contemplates each opticalisomer as well as any combination or ratio of or an optically activeform, for example, a single optically active enantiomer, or anycombination or ratio of enantiomers (e.g., scalemic and racemicmixtures). In addition, the small molecule drug may possess one or moregeometric isomers. With respect to geometric isomers, a composition cancomprise a single geometric isomer or a mixture of two or more geometricisomers.

The present invention also includes pharmaceutical preparationscomprising a compound as provided herein in combination with apharmaceutical excipient. Generally, the compound itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol,myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines, fatty acidsand fatty esters; steroids, such as cholesterol; and chelating agents,such as EDTA, zinc and other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Non-limiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. The optimal amount of any individual excipient isdetermined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (as alyophilizate or precipitate, which can be in the form of a powder orcake), as well as formulations prepared for injection, which are liquidand require the step of reconstituting the dry form of parenteralformulation. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions,normally being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The compounds of the invention can also be administered through the skinusing conventional transdermal patch or other transdermal deliverysystem, wherein the conjugate is contained within a laminated structurethat serves as a drug delivery device to be affixed to the skin. In sucha structure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The compounds of the invention can also be formulated into a suppositoryfor rectal administration. With respect to suppositories, the compoundis mixed with a suppository base material which is (e.g., an excipientthat remains solid at room temperature but softens, melts or dissolvesat body temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the compound (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

In some embodiments of the invention, the compositions comprising thecompounds of the invention may further be incorporated into a suitabledelivery vehicle. Such delivery vehicles may provide controlled and/orcontinuous release of the compounds and may also serve as a targetingmoiety. Non-limiting examples of delivery vehicles include, adjuvants,synthetic adjuvants, microcapsules, microparticles, liposomes, and yeastcell wall particles. Yeast cells walls may be variously processed toselectively remove protein component, glucan, or mannan layers, and arereferred to as whole glucan particles (WGP), yeast beta-glucan mannanparticles (YGMP), yeast glucan particles (YGP), Rhodotorula yeast cellparticles (YCP). Yeast cells such as S. cerevisiae and Rhodotorulaspecies are preferred; however, any yeast cell may be used. These yeastcells exhibit different properties in terms of hydrodynamic volume andalso differ in the target organ where they may release their contents.The methods of manufacture and characterization of these particles aredescribed in U.S. Pat. Nos. 5,741,495, 4,810,646, 4,992,540, 5,028,703,5,607,677 and U.S. Patent Application Publication Nos. 2005/0281781 and2008/0044438.

The invention also provides a method for administering a compound of theinvention as provided herein to a patient suffering from a conditionthat is responsive to treatment with the compound. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe compound (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

In one or more embodiments of the invention, a method is provided, themethod being directed to a method of treating diseases mediated byabnormal protein kinase activity, in particular, receptor tyrosinekinases (RTKs), non-receptor protein tyrosine kinases (CTKs) andserine/threonine protein kinases (STKs), in a patient, in particularhumans, which method comprises administering to said patient apharmaceutical composition comprising a compound of the invention asdescribed herein. Such diseases include, by way of example and notlimitation, cancer, diabetes, hepatic cirrhosis, cardiovascular diseasesuch as atherosclerosis, angiogenesis, immunological disease such asautoimmune disease and renal disease.

In one or more embodiments of the invention, the invention is directedto the use of a compound of the invention as described herein in thepreparation of a medicament which is useful in the treatment of adisease mediated by abnormal PK activity.

The actual dose to be administered will vary depend upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 1000mg, preferably in doses from 0.01 mg/day to 750 mg/day, and morepreferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given compound of the invention (again,preferably provided as part of a pharmaceutical preparation) can beadministered in a variety of dosing schedules depending on the judgmentof the clinician, needs of the patient, and so forth. The specificdosing schedule will be known by those of ordinary skill in the art orcan be determined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Once the clinical endpoint has beenachieved, dosing of the composition is halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents, patent applications, patent publicationsand references identified herein are incorporated by reference in theirentireties. In the event of an inconsistency between the teachings ofthis specification and the art incorporated by reference, the meaning ofthe teachings in this specification shall prevail.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All non-PEG chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

¹H NMR (nuclear magnetic resonance) data was generated by an NMRspectrometer. A list of certain compounds as well as the source of thecompounds is provided below.

Example 1 Synthesis of(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG_(n)-amide

A series of mPEGn-semaxanib conjugates were prepared, each conjugatehaving a different molecule weight. Schematically, the syntheticapproach followed to prepare the conjugates in this series is providedbelow.

For each conjugate in the series, the synthesis was completed in thedark due to the light sensitivity of the materials. A similar syntheticapproach was followed seven times, one for each of mPEG_(n)-NH₂ whereinn=3 through 9. Generally, the(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (300 mg, 1.0 mmol) (obtained commercially) dissolved in 20 mLdimethylformamide (DMF) which resulted in a cloudy yellow solution.Then, triethanolamine (TEA) (276 mg, 2.8 mmol) was added and thesolution turned clear yellow. N-Hydroxybenzotriazole (HOBT) (162 mg, 1.2mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (960 mg,5.0 mmol) were added to the reaction mixture. After all reactants werecompletely dissolved, mPEG_(n)-NH₂ (1.2 mmol) was added. After two hoursthe reaction was complete (checked by HPLC). Then, 150 mL ofdichloromethane (DCM) was added to the reaction mixture. The organicphase was washed with 5% NaHCO₃ (100 mL) and water (150 mL×3). Theorganic phase was dried over sodium sulfate and solvent was removedunder reduced pressure. The yellow oil obtained was purified by Biotageflash chromatography on silica gel (2-8% MeOH in DCM in 23 CV, 40Mcolumn). The desired product was obtained as a yellow solid.(Yield˜38%-66%). The NMR data associated with each synthesis of n=3through 9 for mPEG_(n)-NH₂ is provided below.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₃-amide(“mPEG₃-semaxanib”): ¹H-NMR (500 MHz, CDCl₃)□□ δ 7.95 (s, 1H), 7.35 (s,1H), 7.19 (d, 1H), 6.86 (m, 2H), 6.27 (s, 1H), 3.73-3.65 (m, 10H), 3.54(m, 2H), 3.34 (s, 3H), 2.58 (s, 3H), 2.47 (s, 3H) LC-MS: Calc. 445.2;Found, 446.2 (MH⁺). Molecular weight=445.2.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₄-amide(“mPEG₄-semaxanib”): ¹H-NMR (500 MHz, CDCl₃)□ δ 7.85 (s, 1H), 7.29 (s,1H), 7.15 (d, 1H), 6.80 (m, 2H), 6.26 (s, 1H), 3.75-3.50 (m, 14H), 3.49(m, 2H), 3.30 (s, 3H), 2.47 (s, 3H), 2.36 (s, 3H) LC-MS: Calc. 489.2;Found, 490.2 (MH⁺). Molecular weight=489.2.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₅-amide(“mPEG₅-semaxanib”): ¹H-NMR (500 MHz, CDCl₃) δ 8.15 (s, 1H), 7.24 (s,1H), 7.10 (d, 1H), 6.75 (m, 2H), 6.30 (s, 1H), 3.62-3.56 (m, 18H), 3.53(m, 2H), 3.28 (s, 3H), 2.47 (s, 3H), 2.36 (s, 3H) LC-MS: Calc. 533.2;Found, 534.2 (MH⁺). Molecular weight=533.2.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₆-amide(“mPEG₆-semaxanib”): ¹H-NMR (500 MHz, CDCl₃) δ 8.11 (s, 1H), 7.33 (s,1H), 7.18 (d, 1H), 6.85 (m, 2H), 6.30 (s, 1H), 3.71-3.64 (m, 22H), 3.55(m, 2H), 3.38 (s, 3H), 2.56 (s, 3H), 2.45 (s, 3H) LC-MS: Calc. 577.2;Found, 578.2 (MH⁺). Molecular weight=577.2.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₇-amide(“mPEG₇-semaxanib”): ¹H-NMR (500 MHz, CDCl₃) δ 8.11 (s, 1H), 7.26 (s,1H), 7.15 (d, 1H), 6.77 (m, 2H), 6.26 (s, 1H), 3.63-3.57 (m, 28H), 3.30(s, 3H), 2.49 (s, 3H), 2.38 (s, 3H) LC-MS: Calc. 621.3; Found, 622.4(MH⁺). Molecular weight=621.3.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₈-amide(“mPEG₈-semaxanib”): ¹H-NMR (500 MHz, CDCl₃)□ δ 7.95 (s, 1H), 7.31 (s,1H), 7.15 (d, 1H), 6.77 (m, 2H), 6.25 (s, 1H), 3.61-3.54 (m, 32H), 3.31(s, 3H), 2.50 (s, 3H), 2.41 (s, 3H) LC-MS: Calc. 665.3; Found, 666.4(MH⁺). Molecular weight=665.3.

(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-mPEG₈-amide(“mPEG₉-semaxanib”): ¹H-NMR (500 MHz, CDCl₃)□ 1H) δ 8.11 (s, 1H), 7.32(s, 1H), 7.15 (d, 1H), 6.77 (m, 2H), 6.28 (s, 1H), 3.61-3.54 (m, 36H),3.48 (s, 3H), 2.50 (s, 3H), 2.40 (s, 3H) LC-MS: Calc. 709.3; Found,710.5 (MH⁺). Molecular weight=709.3.

Example 2 In vitro Activity of mPEG_(n)-Semaxanib and Sunitinib

c-Kit tyrosine kinase inhibition mPEGn-semaxanib (prepared as describedin Example 1, n=3-9) and the known tyrosine kinase inhibitor, sunitinib(free base) (obtained commercially as the malate salt, but subsequentlyisolated) were determined.

Using a commercially available screening approach (Caliper Technologies,Mountain View, Calif.), mPEGn-semaxanib (n=3-9) and sunitinib weretested for activity to determine their IC50 values against threetargets. Briefly, the screening approach measures the conversion of afluorescent substrate to a phosphorylated product. The reaction mixture,from a microtiter plate well, is introduced through a capillary sipperonto the chip, where the nonphosphorylated substrate and phosphorylatedproduct are separated by electrophoresis and detected via laser-inducedfluorescence. The signature of the fluorescence signal over time revealsthe extent of the reaction.

Results shown are the averages of replicate wells. Table 1 providesc-Kit results, Table 2 provides VEGFR-2 results and Table 3 provides RETresults. A result of >3E-06 is reported for curves that did not reach50% activity at the highest concentration chosen for the study. Activitymust be ≤50% to report an accurate IC50.

TABLE 1 c-Kit AVG % Activity at Specific Concentration Compound ID3.0E−05 1.0E−05 3.0E−06 1.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09sunitinib malate 0 −1 0 0 6 38 81 91 100 mPEG₃-semaxanib −1 −1 1 2 16 5888 97 98 mPEG₄-semaxamb −1 −1 −1 3 24 70 94 97 93 mPEG₅-semaxamb −1 −1 06 37 76 96 106 99 mPEG₆-semaxamb −1 −1 0 8 44 79 95 94 99 mPEG₇-semaxamb−1 −1 1 11 49 82 96 96 98 mPEG₈-semaxamb −1 −1 1 11 49 75 93 97 98mPEG₉-semaxamb −1 0 2 16 60 90 100 102 105 Compound ID 1.0E−09 3.0E−101.0E−10 IC50 (nM) Fold sunitinib malate 103 100 102 73.00mPEG₃-semaxanib 104 102 100 120.00 1.60 mPEG₄-semaxamb 99 100 97 170.002.30 mPEG₅-semaxamb 100 99 90 230.00 3.10 mPEG₆-semaxamb 97 87 91 290.003.90 mPEG₇-semaxamb 97 96 97 320.00 4.30 mPEG₈-semaxamb 98 94 94 300.004.10 mPEG₉-semaxamb 105 102 95 390.00 5.30

TABLE 2 VEGFR-2 AVG % Activity at Specific Concentration Compound3.0E−05 1.0E−05 3.0E−06 1.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09sunitinib malate 0 −4 −4 1 4 24 62 84 90 mPEG₃-semaxanib −6 −3 −3 0 1647 78 90 90 mPEG₄-semaxanib −6 −4 −4 12 35 56 89 88 96 mPEG₆-semaxanib 0−4 −1 17 34 70 93 91 95 mPEG₆-semaxanib −1 −3 5 31 62 81 94 99 96mPEG₇-semaxanib −6 −3 0 19 46 80 90 95 93 mPEG₈-semaxa−ib −6 −3 2 24 5073 78 85 82 mPEG₉-semaxanib −1 1 8 20 46 61 71 77 84 Compound 1.0E−093.0E−10 1.0E−10 IC₅₀ (nM) Fold sunitinib malate 97 94 90 49.00mPEG₃-semaxanib 87 93 93 110.00 2.24 mPEG₄-semaxanib 92 97 86 180.003.67 mPEG₆-semaxanib 96 90 94 230.00 4.69 mPEG₆-semaxanib 95 85 93520.00 10.61 mPEG₇-semaxanib 95 94 98 310.00 6.33 mPEG₈-semaxa−ib 86 8486 430.00 8.78 mPEG₉-semaxanib 84 84 89 300.00 6.12

TABLE 3 RET AVG % Activity at Specific Concentration Compound 3.0E−051.0E−05 3.0E−06 1.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09sunitinib malate −1 −1 −1 0 7 25 59 83 92 mPEG₃-semaxanib −1 −1 3 12 3868 90 96 95 mPEG₄-semaxanib −1 0 5 22 52 78 95 99 100 mPEG₅-semaxanib −10 6 29 59 84 95 99 102 mPEG₆-semaxanib 0 4 23 48 76 95 95 101 103mPEG₇-semaxanib 0 2 14 38 68 85 93 98 99 mPEG₈-semaxanib 0 5 20 52 75 9294 100 97 mPEG₉-semaxanib 0 5 21 49 74 90 96 105 101 Compound 1.0E−093.0E−10 1.0E−10 IC₅₀ (nM) Fold sunitinib malate 97 98 98 43.00mPEG₃-semaxanib 92 94 92 230.00 5.35 mPEG₄-semaxanib 89 99 100 350.008.14 mPEG₅-semaxanib 98 99 98 440.00 10.23 mPEG₆-semaxanib 101 99 96940.00 21.86 mPEG₇-semaxanib 99 98 98 620.00 14.42 mPEG₈-semaxanib 10299 100 990.00 23.02 mPEG₉-semaxanib 104 97 100 870.00 20.23

As tested by between 100 pM to 10 μM and as shown in the Table 1, thec-Kit activities of mPEG₃₋₉-semaxanib compounds are within about afive-fold of sunitinib. As shown in Table 2, the VEGR-2 activities ofmPEG₃₋₉-semaxanib compounds are within about 10-fold of sunitinib. Asshown in Table 3, the RET activities were lost up to 20-fold ofsunitinib.

Example 3 Synthesis of Ethylene Glycol Linked PEG-Semaxanib Conjugates

Synthesis of(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carbonylchloride (Compound 6): In a 50 mL round-bottomed flask was suspended(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2-one(semaxanib) (0.11 g, 0.47 mmol) in anhydrous THF (5 mL). The suspensionwas transferred to the triphosgene reaction in a second flask.

(Caution: To prevent release of toxic phosgene gas from either thereaction apparatus or rotary evaporator, the equipment setups weresparged through a sodium hydroxide scrub solution via an over pressureor exhaust port.) In a separate 50 mL round-bottomed flask was addedtriphosgene (1.6 g, 5.4 mmol) in anhydrous THF (40 mL) to give acolorless solution. Triethylamine (1.1 mL, 7.8 mmol) was added. Afterten minutes, a semaxanib solution was transferred into this triphosgenesolution. After approximately one hour, the reaction flask was placed onice and cold 4M HC1 solution (30 mL) was added to the flask. The crudeproduct suspension was stirred for ten minutes, filtered and washed withcold 4M HC1 solution (30 mL). The crude product was then placed underhigh vacuum for 18 hours in the presence of P₂O₅. Crude yield (Compound6) was 0.12 g of a red solid. HPLC analysis was on a C18 silica columnapplying an acetonitrile gradient with 0.1% TFA; retention timesobserved were semaxanib 6.2 minutes and carbamoyl chloride product 7.4minutes with ≥33% substitution at 280 nm. The carbamoyl chloride productwas further characterized by reaction with excess n-butylamine(Z)-N-butyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxamideand analyzed by HPLC. HPLC analysis was on a C18 silica column applyingan acetonitrile gradient with 0.1% TFA; retention times observed weresemaxanib 6.2 minutes and butylamine derivative 7.7 min with 81%substitution at 280 nm.

Synthesis of (Z)-(mPEG 20,000)3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate(Compound 6k): In a 50 mL flask was dissolved mPEG-OH 20K (0.5 g, 0.025mmol) in anhydrous toluene. The solvent was evaporated under reducedpressure. The polymer was dissolved in anhydrous DCM (0.5 mL) andpyridine (0.02 mL, 0.23 mmol). To the polymer solution was added asuspension of crude(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carbonylchloride (Compound 6) (23 mg, 0.08 mmol) in anhydrous THF (2.5 mL).After one day, additional Compound 6 (28 mg, 0.1 mmol) was added. After˜3 days, the solvent was evaporated under reduced pressure to a thickoil. The crude product was dissolved in warm anhydrous IPA and slowlycooled to room temperature forming precipitate. The resulting slurry wasfiltered and washed with additional anhydrous IPA. Residual solvent wasevaporated at reduced pressure. Yield was ˜0.45 g of a solid powder.HPLC analysis was on a C18 silica column applying an acetonitrilegradient with 0.1% TFA; retention times observed were semaxanib 4.7minutes and product 4.9 minutes with 96% purity at 280 nm and 59% purityby ELSD. ¹H-NHR (d₆-DMSO): d (ppm) 2.3 (˜3H, s, CH₃); 2.4 (˜3H, s, CH₃);3.2 (˜3H, s, CH₃); 3.6 (˜1800H, bs, PEG backbone); 4.5 (˜2H, s, CH₂);4.6 (<1H, m, OH); 6.1 (˜1H, s, CH); 7.2 (˜2H, m, Ar); 7.7 (˜1H, s, CH);7.8 (˜1H, m, Ar); 7.9 (˜1H, m, Ar); 12.6 (˜1H, s, NH).

Example 4 Pharmacokinetic Study

A pharmacokinetic study of sunitinib and the mPEG₃₋₉-semaxanib compoundsprepared in accordance with Example 1 were evaluated in a standardpharmacokinetic study. Briefly, male Sprague Dawley rats with jugularvein and carotid artery catheters for intravenous dosing and carotidartery catheters only for oral dosing were obtained from Charles RiverLabs. (Wilmington Mass.). The rats were fasted overnight prior todosing. Food was returned after the four-hour timepoint. Each testarticle was assigned a group of three animals and plasma concentrationvalues represent the mean of the values obtained.

For intravenous administration, a one mL syringe was used and theanimals were dosed 2 mg/kg intravenously using the jugular veincatheter. The dead volume of the catheter was flushed with saline toensure the animals received the entire dose. Plasma samples werecollected at 0.03, 0.25, 0.5, 1, 2, 4, 6, 24 and 48 hours post dose.

For oral administration, animals were dosed orally by gavage using a onemL syringe and a gavage needle (18 G×2 inches, Popper & Sons Inc., NewHyde Park, N.Y.). Plasma samples were collected at 0.08, 0.25, 0.5, 1,2, 4, 8, 24 and 48 hours post dose.

All test articles were administered with 0.9% saline and all doses werekept on ice with syringes filled immediately prior to dosing. At the 20mg/mL dosage, the mPEG₃₋₅-semaxanib compounds were not soluble insaline; consequently, the dose for these compounds was diluted to 10mg/mL and the dose volume doubled.

Provided as plots, the mean plasma concentration values for testedarticles after 2 mg/kg intravenous dosing (FIG. 1), after 2 mg/kg oraldosing (FIG. 2), and 20 mg/kg oral dosing (FIG. 3) in rats weredetermined.

What is claimed is:
 1. A compound comprising a semaxanib moietycovalently attached via a spacer moiety to a water-soluble, non-peptidicoligomer.
 2. The compound of claim 1, encompassed by the formula:

wherein: R¹ is either H or halo; R² is selected from the groupconsisting of H, halo, NO₂ and lower alkyl; R³ is either H or loweralkyl; R⁴ is selected from the group consisting of H, halo, lower alkyland CH₂CH₂COOH; R⁵ is selected from the group consisting of H, loweralkyl, COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; R⁶ is selected from the groupconsisting of H, halo, lower alkyl; X is a spacer moiety; and POLY¹ is afirst water-soluble, non-peptidic oligomer; or a pharmaceuticallyacceptable salt thereof.
 3. The compound of claim 1, encompassed by theformula:

wherein: R¹ is either H or halo; R² is selected from the groupconsisting of H, halo, NO₂ and lower alkyl; R³ is either H or loweralkyl; R⁴ is selected from the group consisting of H, halo, lower alkyland CH₂CH₂COOH; R⁵ is selected from the group consisting of H, loweralkyl, COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; R⁶ is selected from the groupconsisting of H, halo, lower alkyl, X is a spacer moiety; and POLY¹ is afirst water-soluble, non-peptidic oligomer; or a pharmaceuticallyacceptable salt thereof.
 4. The compound of claim 1, encompassed by theformula:

wherein: X is a spacer moiety; POLY¹ is a first water-soluble,non-peptidic oligomer; Xr is a releasable linkage-containing spacermoiety; and POLY² is a second water-soluble, non-peptidic oligomer; or apharmaceutically acceptable salt thereof.
 5. The compound of claim 1,encompassed by the formula

wherein: X is spacer moiety; POLY¹ is a first water-soluble,non-peptidic oligomer; Xr is a releasable linkage-containing spacermoiety; and POLY is a water-soluble, non-peptidic oligomer; or apharmaceutically acceptable salt thereof.
 6. The compound of claim 1,wherein the semaxanib moiety is encompassed by the formula:

wherein: R¹ is either H or halo; R² is selected from the groupconsisting of H, halo, NO₂ and lower alkyl; R³ is either H or loweralkyl; R⁴ is selected from the group consisting of H, halo, lower alkyland CH₂CH₂COOH. R⁵ is selected from the group consisting of H, loweralkyl, COOH, CH₂CH₂COOCH₃ and COOCH₂CH₃; and R⁶ is selected from thegroup consisting of H, halo, and lower alkyl.
 7. The compound of claim1, wherein the sexaminib moiety is semaxanib.
 8. The compound of claim1, wherein the water-soluble, non-peptidic oligomer is a poly(alkyleneoxide).
 9. The compound of claim 8, wherein the poly(alkylene oxide) isa poly(ethylene oxide).
 10. The compound of claim 1, whereinwater-soluble, non-peptidic oligomer has from about 1 to about 30monomeric subunits.
 11. The compound of claim 10, wherein thewater-soluble, non-peptidic oligomer has from about 1 to about 10monomeric subunits.
 12. The compound of claim 1, wherein thewater-soluble, non-peptidic oligomer has from about 40 to about 2,000monomeric subunits.
 13. The compound of claim 8, wherein thepoly(alkylene oxide) includes an alkoxy or a hydroxy end-capping moiety.14. The compound of claim 1, wherein a single water-soluble,non-peptidic oligomer is attached to the semaxinib moiety.
 15. Thecompound of claim 1, wherein more than two water-soluble, non-peptidicoligomers are attached to the semaxinib moiety.
 16. The compound ofclaim 1, wherein the semaxanib moiety is covalently attached to thewater-soluble, non-peptidic oligomer via a stable linkage.
 17. Thecompound of claim 1, wherein the semaxanib moiety is covalently attachedto the water-soluble, non-peptidic oligomer via a releasable linkage.18. A composition comprising a compound of claim 1 and apharmaceutically acceptable excipient.
 19. A composition of mattercomprising a compound of claim 1 comprised within a dosage form.
 20. Amethod of treatment comprising administering a compound of claim 1 to asubject in need thereof.